[Prediction of quality markers of Angong Niuhuang Pills based on LC-MS and pull-down with SPR chips].

Elucidating the quality markers(Q-markers) of traditional Chinese medicines is essential for understanding the mechanisms of action and promoting the rational use of traditional Chinese medicines as well as for developing traditional Chinese medicine-derived drugs. Studies have shown that surface plasmon resonance(SPR) is promising in this field. This study proposed a method based on pull-down with SPR chips to predict the Q-markers of Angong Niuhuang pills(AGNHP). Firstly, 71 main chemical components of AGNHP were analyzed by UPLC-Q-TOF-MS, and then network pharmacology was employed to predict the potential targets of AGNHP against stroke. Secondly, the STAT3 protein chip was constructed, and the extract of AGNHP was recovered by pull-down of the SPR system for STAT3 ligand. The potential active ingredients were collected, enriched, and identified as coptisine, palmatine, epiberberine, berberine, worenine, demethyleneberberine, jatrorrhizine, tetrahydrocoptisine, baicalein, and baicalin methyl ester. Next, the affinity constants of the 10 active ingredients were determined as 44.7, 44, 58.1, 51.3, 39.7, 32.1, 49.2, 69.1, 19.7, and 24.9 µmol·L~(-1), respectively. The molecular docking results showed that the 10 compounds could compete for binding with STAT3. This is the first report that SPR combined with UPLC-Q-TOF-MS is reliable and feasible for determining the active ingredients of AGNHP at the molecular level from complex systems. STAT3 could be used as a potential target for the biological quality evaluation of AGNHP.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

OBJECTIVE: To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. METHODS: Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). RESULTS: PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. CONCLUSION: PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Doxorubicin Loaded Dextran-coated Superparamagnetic Iron Oxide Na-noparticles with Sustained Release Property: Intracellular Uptake, Phar-macokinetics and Biodistribution Study.

BACKGROUND: Due to the short biological half-life and serious side effects (especially for heart and kidney), the application of Doxorubicin (Dox) in clinical therapy is strictly limited. To overcome these shortcomings, a novel sustained release formulation of doxorubicin-loaded dextran-coated superparamagnetic iron oxide nanoparticles (Dox-DSPIONs) was prepared. OBJECTIVE: The purpose of this study was to evaluate the intracellular uptake behavior of Dox-DSPIONs and to investigate their pharmacokinetics and biodistribution properties. METHOD: Confocal laser scanning microscopy was employed to study the intracellular uptake and release properties of Dox from Dox-DSPIONs in SMMC-7721 cells. Simple high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was established to study the pharmacokinetics and biodistribution properties of Dox-DSPIONs in vivo after intravenous administration and compared with free Dox. RESULTS: Intracellular uptake experiment indicated that Dox could be released sustainedly from Dox-DSPIONs over time. The pharmacokinetics parameters displayed that the T1/2and AUC0-24h of Dox-DSPIONs were higher than those of free Dox, while the Cmax of Dox-DSPIONs was significantly lower than that of free drug. The biodistribution behaviors of the drug were altered by Dox-DSPIONs in mice, which showed obvious liver targeting, and significantly reduced the distribution of the drug in the heart and kidney. CONCLUSION: Dox-DSPIONs have the sustained-release property in vitro and in vivo, which could significantly prolong blood circulation time, improve bioavailability, and reduce the side effects of Dox. Therefore, the novel formulation of the Dox-DSPIONs has the potential as a promising drug delivery system in cancer therapy.

Inhibition of microRNA-29b suppresses oxidative stress and reduces apoptosis in ischemic stroke.

MicroRNAs (miRNAs) regulate protein expression by antagonizing the translation of mRNAs and are effective regulators of normal nervous system development, function, and disease. MicroRNA-29b (miR-29b) plays a broad and critical role in brain homeostasis. In this study, we tested the function of miR-29b in animal and cell models by inhibiting miR-29b expression. Mouse models of middle cerebral artery occlusion were established using the modified Zea-Longa suture method. Prior to modeling, 50 nmol/kg miR-29b antagomir was injected via the tail vein. MiR-29b expression was found to be abnormally increased in ischemic brain tissue. The inhibition of miR-29b expression decreased the neurological function score and reduced the cerebral infarction volume and cell apoptosis. In addition, the inhibition of miR-29b significantly decreased the malondialdehyde level, increased superoxide dismutase activity, and Bcl-2 expression, and inhibited Bax and Caspase3 expression. PC12 cells were treated with glutamate for 12 hours to establish in vitro cell models of ischemic stroke and then treated with the miR-29 antagomir for 48 hours. The results revealed that miR-29b inhibition in PC12 cells increased Bcl-2 expression and inhibited cell apoptosis and oxidative damage. These findings suggest that the inhibition of miR-29b inhibits oxidative stress and cell apoptosis in ischemic stroke, producing therapeutic effects in ischemic stroke. This study was approved by the Laboratory Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University (approval No. 201709276S) on September 27, 2017.

Synthesis and Characterization of Curcumin-Functionalized HP-ß-CD-Modified GoldMag Nanoparticles as Drug Delivery Agents.

Curcumin, a polyphenol extracted from turmeric (Curcuma longa), has emerged as a potent multimodal cancer-preventing agent. It may attenuate the spread of cancer and render chemotherapy more effective. However, curcumin is neither well absorbed nor well retained in the blood, resulting in low efficacy. In an attempt to enhance the potency and to improve the bioavailability of curcumin, new delivery agents, hydroxypropyl-beta-cyclodextrin (HP-ß-CD)-modified GoldMag nanoparticles (CD-GMNs) were designed and synthesized to incorporate curcumin. The CD-GMNs were characterized by Fourier Transform Infrared Spectroscopy (FT-IR), Thermo-gravimetric Analysis (TGA), X-ray Diffraction (XRD), Dynamic Light Scattering measurements (DLS), Transmission Electron Microscopy (TEM) and Vibrating Sample Magnetometer (VSM) analyses. For the magnetic carrier of CD-GMNs, the content of HP-ß-CD was 26.9 wt%. CD-GMNs have a saturation magnetization of 22.7 emu/g with an average hydrodynamic diameter of 80 nm. The curcumin loading, encapsulation efficiency and releasing properties in vitro were also investigated. The results showed that the drug encapsulation ratio was 88% and the maximum curcumin loading capacity of CD-GMNs was 660 µg/5 mg. In vitro drug release studies showed a controlled and pH-sensitive curcumin release over a period of one week. Collectively, our data suggest that HP-ß-CD-modified GoldMag nanoparticles can be considered to form a promising delivery system for curcumin to tumor sites. Targeting can be achieved by the combined effects of the application of an external magnetic field and the effect on drug release of lower pH values often found in the tumor microenvironment.

Mitochondrial tRNAArg T10454C variant may not influence the clinical expression of deafness associated 12S rRNA A1555G mutation.

In this study, we examined the "pathogenic" role of the T10454C mutation in mitochondrial tRNA(Arg) gene in deafness expression as increasing reports provided an active role of this mutation in clinical manifestation of deafness associated 12S rRNA A1555G mutation. For this purpose, we reanalyzed the complete mitochondrial DNA (mtDNA) sequence data containing the T10454C mutation. Moreover, we analyzed the reported "polymorphisms" of mtDNA in the proband using the phylogentic approach. To our surprise, other mutations which occurred at protein-coding genes played more important roles in resulting mitochondrial dysfunctions by using the bioinformatic tool. In addition, evolutionary conservation analysis of the T10454C mutation indicated that this mutation was not conserved between different species. To our knowledge, this is the first report that the T10454C variant may not modulate the phenotypic expression of the deafness associated A1555G mutation.

Gold magnetic nanoparticle conjugate-based lateral flow assay for the detection of IgM class antibodies related to TORCH infections.

In this study, a lateral flow immunochromatographic assay (LFIA) system for the detection of immunoglobulin M (IgM) antibodies, related to TORCH [(T)oxoplasmosis, (O)ther agents, (R)ubella (also known as German Measles), (C)ytomegalovirus, and (H)erpes simplex virus infections], based on gold magnetic nanoparticles, was established. Following modification with poly(methacrylic acid), the gold magnetic nanoparticles conjugated with an anti­human IgM antibody (µ­chain specific) to construct a probe. A lateral flow assay device was constructed based on these conjugates. IgM antibodies to four types of pathogens, notably toxoplasmosis, rubella virus, cytomegalovirus and herpes simplex virus type 2, were detected using this device. Compared with commercial colloidal gold­based LFIA strips, our method exhibited higher sensitivity. No interference with triglycerides, hemoglobin and bilirubin occurred, and no cross­reactivity was noted among the four pathogens. The gold magnetic nanoparticle­LFIA strips were used to assess 41 seropositive and 121 seronegative serum samples. The sensitivity was 100% (162/162) and the specificity was 100% (162/162). This method cannot only be used for the detection of TORCH IgM-specific antibodies, but it can potentially be developed for use in the diagnosis of other acute or recently identified autoimmune diseases.

Dextran-coated superparamagnetic nanoparticles as potential cancer drug carriers in vivo.

Dextran-coated superparamagnetic iron oxide nanoparticles (DSPIONs) have gained considerable interest, because of their biocompatibility and biosafety in clinics. Doxorubicin (Dox), a widely used chemotherapeutic drug, always has limited applications in clinical therapy due to its serious side effects of dose-limiting irreversible cardiotoxicity and myelo suppression. Herein, DSPIONs were synthesized and developed as magnetic carriers for doxorubicin. The Dox-DSPION conjugates were evaluated in the in vitro test of Dox release, which showed pH-dependence with the highest release percentage of 50.3% at pH 5.0 and the lowest release percentage of 11.8% in a physiological environment. The cytotoxicity of DSPIONs and Dox-DSPIONs evaluated by the MTT assay indicated that DSPIONs had no cytotoxicity and the conjugates had significantly reduced the toxicity (IC50 = 1.36 µg mL(-1)) compared to free Dox (IC50 = 0.533 µg mL(-1)). Furthermore, confocal microscopic data of cell uptake suggest that less cytotoxicity of Dox-DSPIONs may be attributed to the cellular internalization of the conjugates and sustainable release of Dox from the formulation in the cytoplasm. More importantly, the results from the rabbit VX2 liver tumor model test under an external magnetic field showed that the conjugates had approximately twice the anti-tumor activity and two and a half times the animal survival rate, respectively, compared to free Dox. Collectively, our data have demonstrated that Dox-DSPIONs have less toxicity with better antitumor effectiveness in in vitro and in vivo applications, suggesting that the conjugates have potential to be developed into chemo-therapeutic formulations.

The effects of antiepileptic drug valproic acid on apoptosis of hippocampal neurons in epileptic rats.

This study activated chronic epilepsy rat model with PTZ (Pentetrazole) and controlled epilepsy with VPA (valproate). By doing that, we detected the apoptotic cells of hippocampus and figured out the expression variation of hippocampal neurons applying immunohistochemical methods. We selected 30 adolescent male SD (Sprague-Dawley) rats (201±29g), which were clean and healthy. The rats were divided into three groups and 10 were in each. Then we detected the apoptotic cells of hippocampus using TUNEL (Terminal Deoxynucleotidyl Transferase Mediated Biotinylated Deoxyuridine Triphosphate Nickel End Labeling Technique) and Bcl-2 and Bax positive cells applying immunohistochemical methods. The results showed Bcl-2 positive cells of hippocampal neuron in VPA group had no statistical variation as compared with PTZ group; Bax positive cells of hippocampal neuron in VPA group induced as compared with PTZ group; dye and density of the positive cells were both decreased, whereas there was no statistical variation when compared with normal control group. Based on the experiment, we reached the conclusion that, VPA showed no harm to epileptic rats and the hippocampal neuronal apoptosis after epilepsy and anti-epileptic drug was probably resulted from the decrease of Bcl-2 expression and increase of Bax expression.

Functional polymorphism in exon 5 and variant haplotype of the interleukin-1 receptor-associated kinase 1 gene are associated with susceptibility to and severity of sepsis in the Chinese population.

BACKGROUND: The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is believed to play an important role in the pathogenesis of sepsis. Recent studies have suggested that the IRAK1 functional genetic variant could affect the severity of sepsis in Caucasians. In this report, we have investigated whether polymorphisms at the IRAK1 gene are associated with the susceptibility to and severity of sepsis among the Chinese population. METHODS: Haplotype-tagging single nucleotide polymorphisms (htSNPs) were selected from the HapMap database. They were genotyped in 255 patients with sepsis and 260 control subjects by PCR/restriction fragment length polymorphism (RFLP) analysis. The association between the selected htSNPs and the susceptibility to and severity of sepsis were estimated by Logistic regression with adjustments for age, sex, smoking, drinking, chronic disease status, Acute Physiology and Chronic Health Evaluation (APACHE) II score and primary diseases. RESULTS: rs1059702 was selected to represent the six linked htSNPs for IRAK1. Genotype frequencies of the htSNPs were in Hardy-Weinberg equilibrium for females, as were allele frequencies for both sex groups. Associations were observed in females between the htSNPs C/C genotype and increased susceptibility to sepsis (odds ratio (OR), 5.46; 95% confidence interval (CI), 1.12 - 26.67; P = 0.018), and such associations were also observed between the IRAK1 variant haplotype (CC/C-allele) and increased susceptibility to sepsis (OR, 1.68; 95% CI, 1.05 - 2.70; P = 0.031) when compared with the T/T + T/C genotype and the wild-type haplotype (TC + TT/T-allele). In the multiple organ dysfunction syndrome (MODS) subgroup, the variant haplotype was also associated with increased severity of sepsis (OR, 2.37; 95% CI, 1.13 - 4.94; P = 0.02) when compared with the wild haplotype. This association was not significant in male patients. CONCLUSIONS: The functional polymorphism in exon 5 and the variant haplotype of IRAK1 gene mediate susceptibility to and severity of sepsis. IRAK1 might be a genetic risk factor for the occurrence and development of sepsis in the Chinese population.